Individuals with human immunodeficiency virus (HIV) experience brain pathology associated with greater motor/mood/cognitive disorders (collectively termed neuroAIDS) than the general population and these effects are exacerbated among opiate abusers. Moreover, gender differences exist with opiate-abusing women at greater risk to contract HIV compared to opiate-abusing men; yet, some women may experience lesser neuroAIDS symptomology than men once infection occurs. We recapitulated these effects in mice and have begun to elucidate the sex steroid hormones that interact with opiates to confer protection to neuroAIDS. The present proposal will begin to reveal the effects and mechanisms of sex steroid interactions with opiates and HIV in a translational approach that utilizes cultured murine and human neural cells, as well as transgenic whole-animal murine models. The goal of this Pathway to Independence Award (K99/R00) is to provide training to Dr. Jason Paris to discern the cellular/molecular mechanisms by which sex steroid milieu may influence opiate/HIV interactions for neuroAIDS pathology. Dr. Paris will receive advanced training in murine and human neural cell culture and imaging techniques for detection of sub lethal neuronal pathogenicity, intracellular ions, and mitochondrial membrane integrity. In the K99 training phase, Dr. Paris will work under the tutelage of Dr. Kurt Hauser (Primary Mentor) to examine the potentially-protective effects that sex steroids may exert over synergistic actions between opiates and the HIV-1 virotoxins, Tat (Aim 1a) or gp120 (Aim 1b) in murine neural co-cultures. Viable/degenerating neurons, astrocytes, and microglia will be detected via immunocytochemistry, biocytin-filled striatal medium spiny neurons (MSNs) will be assessed for sub lethal changes in morphology, and supernatants will be assessed for production of cytokines/ chemokines, reactive oxygen species, and nitrites. In murine (Aims 2a and 2b) and human (Aim 2c) neural cultures, steroid protection against opiate interactions with Tat (2a), gp120 (2b), or X4-/R5-tropic HIV (2c) will be assessed for effects on MSN morphology, ion imaging for [Ca] i and [Na] i within MSN soma and dendrites, 2+ + and mitochondrial membrane destabilization (via Rhodamine 123 fluorescence imaging). In the R00 independent phase, Dr. Paris will examine the protective effects of central steroid formation on opiate/HIV protein interactions in vivo using whole-animal models that conditionally-express central HIV-1 Tat (Aim 3a) or constitutively-express central gp120 (Aim 3b). Male and female mice will be assessed for neuroAIDS-like motor, affective, and cognitive behavior. Striatal tissue will be assessed for endpoints described in Aim 1. Lastly, the influence of pharmacodynamic targets that overlap between steroids, opiates, and HIV proteins will be examined in murine neural co-cultures obtained from Tat- or gp120-transgenic mice (Aim 3c). Cultures will be pretreated with pharmacological antagonists and examined as in Aims 1 and 2. This proposal is timely, provides novel training, and will enable the development of an innovative and independent research program.